Creation Of A Rapid Detection Kit For Transgene Labeled HPH And BAR Colloidal Gold

Hygromycin-B-phosphotrans ferase(hph)and bislaphos resistance(bar)genes are ideal screening markers for transgenic plants,with the greatest advantages of high selection efficiency,stable traits of transgenic crops and little or no toxicity to transformed cells.With the extensive promotion of genetically modified plants,the traditional methods for detecting genetically modified products are time-consuming and laborious,require professional instruments,and require high personnel requirements.Therefore,there is an urgent need to create a detection method with high sensitivity,faster detection time and lower cost for laboratory transgenic research and development and on-site detection of transgenic plants.In this study,the HPH and BAR genes were analyzed by bioinformatics technology,the rare codons of six amino acids were optimized,the prokaryotic expression vector was constructed and the recombinant protein was successfully induced.Inducible protein was purified by urea dissolution and then desalted and renatured by gel chromatography.New Zealand white rabbits were immunized with renatured proteins separately.The antibody titer was higher after four times of booster immunization.The purified antibody was confirmed to have good specificity by Western blot.A rapid detection kit was created based on colloidal gold immunochromatography technology,and the performance of the kit was evaluated and optimized.The main findings are as follows:1.HPH and BAR genes were analyzed by bioinformatics technology,and 32 codons were optimized to improve the expression efficiency of arginine,glycine,isoleucine,proline,leucine and threonine in E.coli.The optimized HPH and BAR sequences were linked to the pET-30a vector.The constructed prokaryotic expression vector was transformed into E.coli BL21(DE3)and induced by IPTG.Electrophoretic analysis showed that the induced recombinant protein size was 48 kD and 25 kD.The two proteins were further purified by nickel ion affinity chromatography,with Hph protein concentration of 1248.6 μg/mL and Bar protein concentration of 1208 μg/mL.2.The purified proteins were renatured by dialysis and gel chromatography,respectively.The results showed that the renaturation proteinurea by dialysis was not completely removed,the protein loss was too large,while the urea by gel chromatography was thoroughly removed,and the protein loss was low.Therefore,gel chromatography was used for salt removal renaturation.3.New Zealand white rabbits were immunized with Hph and Bar protein after renaturation by five-point injection,with the immunization dose of 400 μg.The Hph antiserum titer after primary immunization was 1:102,400 and the Bar antiserum titer was 1:51,200.The titer of Hph antibody was 1:1,638,400 and that of Bar antibody was 1:3,276,800 after four booster immunizations.The rabbits were killed and plasma was removed,and serum antibodies and blood cells were isolated.The antibodies purified by saturated ammonium sulfate method were detected by Western blot.The results showed that the two antibodies prepared had good specificity.4.The most suitable amount of 1%chloroauric acid added to 1.5 mL of 1%trisodium citrate in the preparation of colloidal gold was determined,and the diameter of the prepared particles was about 28.5 nm colloidal gold solution,the prepared solution presents a bright and transparent wine red as a whole.According to the results of ultraviolet and visible light scanning,the main peak width is smaller,the absorption width of the visible region is narrow,and the size of gold particles is uniform,indicating that the prepared colloidal gold is of good quality and can be used for subsequent tests.5.The relative amount of K2CO3 is used to determine the optimal pH when colloidal gold labels Bar antibodies.The results showed that the gold particles in 1 mL colloidal gold solution with 4 μL of 0.1 mol/L K2CO3 could stably bind to the antibody.The optimum concentration of colloidal gold and Bar antibody binding was determined to be 1 mL.The optimum concentration of colloidal gold added with 12 μg antibody and kit detection line spray point antibody was 1.0 mg/mL.6.The minimum detection limit of the developed BAR rapid detection kit is 1μg/mL,and there is no cross-reaction with Hph protein and BSA protein,indicating that the kit has good specificity.The acceleration loss of the simulated kit was 30 d at 37℃,and the detection limit did not change significantly,indicating that the kit had good thermal stability.By comparing with the sensitivity of the commercially available Bar rapid detection kit,the kit created in this study has shorter developing time,more obvious color and lower detection limit when detecting positive samples.7.HPH rapid detection kit has established a colloidal gold rapid detection system,but there has always been a false positive interference in the test.In the later stage,it is still necessary to optimize the process of the kit and thoroughly solve the problem of false positive before it can be applied to the market.